The immune response of CD8+ T lymphocytes to protein antigens can be detected only with a great expenditure using known methods. It depends on the presentation of the epitopes derived from these antigens on MHC class I molecules on cells and can be measured through measuring a cytotoxic response induced by exposure. This experimental set-up is usual and takes one to several weeks in which the CD8+ T lymphocytes must be stimulated with the antigen in a suitable cell culture and are then incubated in a cytotoxicity test with suitable target cells which have been loaded with peptides from this antigen or transfected with the antigen or parts thereof. The induction of a response of the CD8+ T lymphocytes is measured from the degree of destruction of target cells, which requires suitable controls and includes a great experimental and time expenditure.
The detection of the immune response of CD4+ T lymphocytes to protein antigens is somewhat less complicated. The response of CD4+ T lymphocytes to protein antigens depends on the presentation of the epitopes derived from these antigens on MHC class II molecules on cells and can be measured through the proliferation of such cells in the presence of the antigen or upon exposure to this antigen, e.g., through the incorporation of tritiated thymidine. This experimental set-up is usual and takes several days up to a week or longer. The presence of a CD4+ T lymphocyte response to protein antigens can further be measured in a known method in which a suspension containing CD4+ T lymphocytes is incubated with the corresponding protein followed by detecting the CD4+ T lymphocyte induction through the presence of intracellular cytokines by flow cytometry.
The presence of a CD8+ T lymphocyte or CD4+ T lymphocyte response to individual epitopes can further be measured in a known method in which a suspension containing CD8+ and/or CD4+ T lymphocytes is incubated with peptides from this protein followed by detecting the CD8+ or CD4+ T lymphocyte induction through the presence of intracellular cytokines by flow cytometry, making use of the fact that peptides can be charged directly from outside onto the MHC class I or MHC class II molecules on cells, circumventing intracellular processing. In this method, it can be achieved by a suitable grouping of peptides that stimulating peptides can be identified and thus epitopes can be determined. The grouping used in this way distributes all possible epitopes to several, and mostly a large number of, runs so that it can be established whether individual peptides from this protein can induce a T lymphocyte response and it can be established which of the peptides occurring in the individual groups have led to such stimulation (this is described in F. Kern et al., Journal of Virology, October 1999, p. 8179-8184, and in WO 99/36568).
However, this grouping allows neither to determine systematically in a single measurement with a corresponding control whether a T lymphocyte response against the protein is present at all, nor to tell how strong the response (the proportion of the reactive lymphocytes in percent of the total CD8+ or CD4+ T lymphocytes) to this protein is all in all. To do this, the usual grouping in this method for the identification of epitopes would require several stimulation and measuring runs, depending on the number of peptides used. The application described in the literature aims at the precise identification of epitopes and therefore uses groups of peptides whose size is chosen in such a way that as few as possible individual peptides must be tested to establish the stimulating activity of a peptide group. However, the smaller the group size is chosen, the more groups have to be tested. Therefore, as the most favorable variant, a number of groups is chosen in this method which is twice the square root of the next square number exceeding the number of the peptides (unless the number of peptides is itself a square number).
This may be exemplified by the pp65 protein of the human cytomegalovirus. 138 peptides were synthesized which cover the amino acid sequence of the whole protein (561 amino acids) to the full length thereof, neighboring peptides overlapping by 9 amino acids each. 138 is not a square number. The next higher square number from 138 is 144 (12×12). Thus, the peptides were distributed to 2×12, i.e. 24, groups in such a way that each peptide occurs in exactly two different groups. By combining the groups with positive results (stimulation), the stimulating peptide can be concluded directly (when only two groups show a positive result), or it can be narrowed down to a small number of candidate peptides which can be retested individually, if more then two groups of peptides have resulted in positive stimulation results. The principle of this grouping has been described in some detail by F. Kern et al., Journal of Virology, October 1999, p. 8179-8184. A possibility for telling by one single run with a corresponding negative control whether a protein has a stimulating effect on CD8+ T lymphocytes, i.e., whether the amino acid sequence of this protein contains epitopes which are recognized by CD8+ T lymphocytes, has not been described to date.